Materials and methods Directory UMM :Data Elmu:jurnal:B:Brain Research:Vol888.Issue2.2001:

G . Costa et al. Brain Research 888 2001 336 –342 337 nists provided evidence for the specific mediation by in aCSF containing 0.2 ascorbic acid. Nicotine and CHL different subtypes of nAChR [29,31,43]. were dissolved in saline. In spite of this in vitro evidence, in vivo studies are still controversial when regarding the neuroprotective prop- 2.3. Intranigral injection of 6-OHDA erties of nicotine treatments. While continuous nicotine infusion has been demonstrated to protect against neuronal Animals were anaesthetized with halothane Fluothane, loss produced by dopamine pathways hemitransection Zeneca and placed in a D. Kopf stereotaxic frame. [25,26], the striatal depletion of dopamine after injection of Through a skull hole, the needle 0.022 mm o.d., 0.013 6-hydroxydopamine 6-OHDA in the substantia nigra mm i.d. of a Hamilton syringe 5 ml, attached to a SN was unaltered by the same treatment [10]. 1-Methyl- micro-injection unit D. Kopf, was lowered to the SN. 4-phenyl-1,2,3,6-tetrahydropyridine MPTP systemic ap- Coordinates H, 24.8; L, 22.2; V, 27.2 were determined plication in vivo resulted in a significant decrease of from bregma, according to the atlas of Paxinos and Watson striatal dopamine content that was not prevented by [37]. nicotine in some studies [21], while another group showed A total of 2.0 ml of a 6-OHDA solution 3 and 5 mg ml that nicotine had protective effects against diethyldithio- for the 6- and 10-mg doses was injected for 4 min and the carbamate enhancement of MPTP lesions [34]. Moreover, needle was slowly withdrawn, allowing the drug to diffuse in the MPTP model of experimental parkinsonism, nicotine for another minute. Body temperature was maintained at has even shown an enhancement of the neurotoxicity 378C using a temperature control system. [5,19]. Beyond these discrepancies, it is likely that the great 2.4. Experimental groups and nicotine administration variety of experimental conditions reported, differing in schedule the schedule and method of nicotine administration, may in part explain the differences observed. Thus, reports using 2.4.1. Partial lesion 6 mg 6-OHDA in the SN chronic treatments did not show effects in vivo [21,27], Five groups of rats n58 injected with 6-OHDA 6 mg while the acute intermittent administration appeared to in the right SN, received 1 mg kg nicotine subcutaneously show protective effects in the models reported [27,34]. according to the following protocols: 1 nicotine 18 h In this context, and as a contribution to the characteriza- before 6-OHDA; 2 nicotine 4 h before 6-OHDA; 3 tion of the role played by nAChR in Parkinson’s disease, nicotine 4 h before, and 20, 44 and 68 h after 6-OHDA; 4 we studied the putative neuroprotective effects of nicotine nicotine 20, 44, 68 h after 6-OHDA; 5 similar adminis- in the 6-OHDA model of experimental parkinsonism, tration schedule to 3 plus CHL 10 mg kg s.c. 30 min assessing whether the timing and schedule of nicotine before the first application of nicotine. administration as well as the extent of the lesion are factors Each of the five experimental groups had its own control that could effectively influence the neuroprotection profile. group of 6-OHDA plus saline instead of nicotine. For control of 6-OHDA vehicle, six rats were injected with similar volumes of aCFS with 0.2 ascorbic acid.

2. Materials and methods

2.4.2. Extensive lesion 10 mg 6-OHDA in the SN Animals injected with 10 mg 6-OHDA received nicotine 2.1. Animals or saline treatment according to the administration schedule 3. Experiments were carried out using male Sprague–Daw- ley rats 230–260 g. Animals had access to food and 2.5. Neurochemical analysis water ad libitum, and were housed in groups of six in a temperature controlled environment on a 12-h light dark For neurochemical analysis rats were decapitated 8 days cycle. after 6-OHDA injection, brains rapidly removed and the left and right SN and corpus striatum CS dissected out 2.2. Drugs and reagents and kept at 2708C. Next day tissue samples were weighed, sonicated in perchloric acid 0.1 M 200 and 1000 ml for Chemicals for HPLC analysis, artificial cerebrospinal the SN and CS, respectively and centrifuged 15 0003g fluid aCSF and saline were purchased from Baker for 15 min at 48C. Then, samples were injected into an Phillipsburg, PA, USA. Dopamine hydrochloride, 3,4- HPLC system PM-80 BAS, USA equipped with a C-18 dihydroxyphenylacetic acid DOPAC, 6-OHDA, 2-nico- column 5-mm particles, 220 mm34.6 mm; BAS, USA tine tartrate and L -ascorbic acid were obtained from and a electrochemical detector LC-4C BAS with oxida- Sigma St. Louis, MO, USA. Chlorisondamine CHL was tion potential at 10.75 V glassy working carbon electrode donated by Novartis Pharmaceuticals NJ, USA. 6-OHDA versus an Ag AgCl reference electrode. The mobile phase solution for intranigral injection was prepared dissolving it was composed of citric acid 0.15 M, sodium octyl 338 G sulphate 0.6 mM, 4 acetonitrile and 1.6 tetrahydro- furan at pH 3.0; with a flow rate of 1.0 ml min. 2.6. Statistical analysis Dopamine is presented as percent of right versus left tissue levels, considering the left side untreated as 100. In each experimental group comparison were made against controls as defined in Section 2.4. For turnover analysis, in each experimental group, the ratio DOPAC DA of the ipsilateral injected side was compared with its control contralateral side. Means comparison was performed Fig. 1. Striatal dopamine assessed 8 days after the injection of 6-OHDA using independent t-test two-tailed. Statistical signifi- 6 mg expressed as percent mean1S.D. of right versus left CS. cance was chosen at P,0.05. Subcutaneous administration of nicotine 4 h before, and 20, 44 and 68 h after 6-OHDA injection counteracted significantly the dopamine decrease 6-OHDA1nicotine versus 6-OHDA1saline; P,0,05. The same ad- ministration schedule of nicotine did not prevent the dopamine decrease

3. Results